Protein binding or protein-protein interactions can be broadly defined as the discrete interaction of the surface of one protein with the surface of another protein. Such discrete interaction arises when residues of one protein are proximally located to residues of another protein and attractive forces between the residues such as vander Waals forces, ionic bonds and hydrogen bonds exist. Specific protein-protein interactions which occur in higher living organisms include but are not limited to those in which involve: a receptor-binding protein binding to a receptor; a pathogen antigen binding to a host cell receptor; protein interactions at cellular attachment sites; and, adhesion proteins interactions.
Examples of receptor-binding proteins, hereinafter also referred to as ligands, include cytokines, hormones and growth factors. These proteins bind to receptors on cells and cause changes in cellular activity or function. For example, cytokines are a variety of proteins which are cellular messengers, each cytokine having a specific effect upon a cell. Likewise, hormones and growth factors are also messengers which affect the function and activity of cells.
Pathogens are infectious organisms, such as bacteria, fungi, parasites, and viruses and, additionally, neoplasms, all of which express specific antigens. Such typically, there are specific sites on antigens, hereinafter referred to as binding epitopes or epitopes, which bind to a complementary portion of a cellular protein called a receptor site.
A great deal of effort has been expended in search of compounds which specifically either simulate, that is mimic, or block protein-protein interactions in cells.
With respect to cytokines, hormones and growth factors, a great deal of effort has been made to purify the natural proteins from natural sources or to synthetically produce them by chemical means or using recombinant DNA technology. While some success has been achieved, these molecules are quite large, difficult to handle and expensive to obtain. A great deal of effort is also directed at discovering synthetic ligands which either mimic the activity of natural proteins or which block the activity of natural proteins. Blocking natural protein activity can be achieved by either competing for the receptor with an inactive ligand (antagonist) or by having an agent bind to the natural protein and thereby prevent it from binding to the receptor.
There is a need for synthetic peptides and/proteins which mimic the activity of the natural biologically active proteins which interact with receptors. Such mimicking molecules would be useful as agents to affect the cells in the same way as the natural protein. Likewise, the discovery of antagonists, that is, molecules which block the receptor without having an effect on cellular function or activity would be useful. Furthermore, the discovery of agents which specifically interact with biologically active proteins and thereby render them unable to bind to receptors is also desirable. Molecules that prevent binding by a natural biologically active protein to its receptor in cases where the natural protein is believed to an agent associated with a disease condition or disorder are useful as drugs for preventing or treating such disease conditions or disorders.
A great deal of knowledge has been developed in the field of immunology, including at the molecular level. Advances in molecular biology have indicated that immunoglobulins, major histocompatibility complex antigens and T-cell receptors are all members of a family of molecules referred to as the immunoglobulin superfamily. During evolution, it is likely that a single, useful gene duplicated, and its copies diverged to create related molecules with distinct functions. Accordingly, immunoglobulins, which are agents of humoral immunity; T-cell receptors, which are associated with humoral as well as cellular immunity; and major histocompatibility complex molecules, involved in antigen presentation and the discrimination between self and nonself, all share homologies inherited from their common ancestor and exhibit related biological functions.
Of the members of the superfamily, the structure and function of immunoglobulins is best understood. Immunoglobulin molecules consist of a constant region and a variable region. The constant region is associated with cellular effector functions whereas the variable region participates in antigen recognition and binding.
Immunoglobulins of the most common class, IgG, consist of two heavy chains and two light chains linked together by noncovalent associations and also by covalent disulfide bonds. Each of the chains possesses a constant as well as a variable region. In the immunoglobulin molecule, the variable region is subdivided into framework regions, which are similar in structure among immunoglobulins, and hypervariable, complementarity determining regions (CDRs) which participate directly in antigen binding in the immunoglobulin active site.
X-ray crystallographic studies of purified immunoglobulin molecules have indicated that the active site is a crevice formed by the heavy and light chain variable regions, and that the dimensions of the active sites vary among immunoglobulin molecules consequent to amino acid sequence variations (Hood et al., 1978, in "Immunology," The Benjamin/Cummings publishing Co., Inc., Menlo Park, p. 208). Amino acid sequence, crystallographic structure, and specially designed hapten probes have been used in conjunction with computer analysis to elucidate the relationship between an immunoglobulin and the antigen which it recognizes.
Pathogens generally express antigens which are recognized by host immune systems as foreign and become the target of an immunological response to eliminate the infectious pathogen. Pathogen antigens often bind to cellular receptors on a host's cells as part of the process of infection of the host by the pathogen. In order to immunize the host and reduce the effectiveness of the pathogen to mount a challenge to the host, a number of vaccination strategies have been devised.
Several strategies have been employed to develop safe, effective vaccines against viral and bacterial pathogens. At present most vaccines in use consist of live attenuated pathogens, killed pathogens, components of a pathogen, or modified toxins (toxoids). See Institute of Medicine, "Vaccine Supply and Innovation", Washington, D.C.: National Academy Press (1985). While these preparations have been successfully used for many infectious diseases, many pathogens exist where these approaches have not worked or have not been applicable. Certain pathogens are potentially too dangerous to contemplate the use of attenuated or even inactivated preparations. The risk of developing cancer from immunization with certain retroviruses, or of developing acquired immunodeficiency syndrome (AIDS) from immunization with human immunodeficiency virus (HIV) underscores the drawbacks associated with the use of whole virus preparations for vaccination. In addition many pathogens display a marked antigenic heterogeneity that makes effective vaccination difficult. These considerations have led us to seek alternative method for effective immunization.
The idiotype network theory of N. K. Jerne, Ann. Immunol. (Paris) 125: 337-389, (1974), implies that an anti-idiotypic antibody raised against a neutralizing antibody specific for a pathogen would mimic that pathogen immunologically. Immunization with the anti-idiotype should result in the development of a significant anti-pathogen response with the elicitation of neutralizing antibodies and cell-mediated immunity. In recent years there have been several examples where this strategy has been effective, including reovirus type 3. See Sharpe, A. H., et al., J. Exp. Med. 160: 195-205 (1984); Kauffman, R. S., et al., J. Immunol., 131: 2539-2541, (1983); and Gaulton, G. N., et al., J. Immunol. 137: 2930-2936. With respect to Sendai virus, see Ertl, H. C. and Finberg, R. W., Proc. Natl. Acad. Sci. USA 81: 2850-2854 (1984). For report relating to rabies see Reagen, K. J. et al., J. Virol. 48: 660-666 (1983). This approach has been discussed in connection with polio virus in Uydeltaag, F.G.C.M. and Osterhaus, A.D.M.E., J. Immunol. 134: 1225-1229 (1985).
One of the key aspects of this approach is that a portion of the anti-idiotype mimics a portion of the pathogen antigen and induces a neutralizing response. Thus a potent anti-idiotype vaccine would seem to be an ideal immunogen in cases where intact pathogen could not be used or where irrelevant non-neutralizing epitopes dominate the immune response. However, the practical application of anti-idiotypes as vaccine has been limited by the difficulties in making human monoclonal antibodies and in the danger of producing serum sickness by using xenogeneic antibodies.
Another method currently under intensive investigation is the use of synthetic peptides corresponding to segments of the proteins from pathogenic microorganisms against which an immune response is directed. This approach has been successful in several instances including feline leukemia virus (Elder, J. H. et al., J. Virol. 61: 8-15, 1987), hepatitis B (Gerin, J. L., et al., Proc. Natl. Acad. Sci. USA, 80: 2365-2369 1983), Plasmodium falciparum (Cheung, A., et al., Proc. Natl. Acad. Sci. USA 83: 8328-8332, 1986), cholera toxin (Jacob, C. O., et al., Eur. J. Immunol. 16: 1057-1062, 1986) and others. When these peptides are capable of eliciting a neutralizing immune response they appear to be ideal immunogens. They elicit a specific response and typically do not lead to deleterious effects on the host. However, it can be difficult to predict which peptide fragments will be immunogenic and lead to the development of a neutralizing response.
It would be desirable to develop immunogens that elicit a response to specific neutralizing epitopes without causing responses to extraneous epitopes that could "dilute" the specific response or lead to harmful immune complex formation.
The present invention relates to a method of identifying specific linear and constrained discrete portions of a biologically active proteins involved in protein-protein interactions. By identifying such specific and discrete portions, biologically active peptides can be constructed which mimic the biological activity of the biologically active protein or which block the activity of the biologically active protein. Thus, biologically active peptides can be constructed which act as ligands that act on mammalian cells by binding to the receptor sites of those cells to alter or affect their function or behavior, or to prevent the binding of the natural biologically active protein to the cellular receptor, thereby preventing the biologically active protein from affecting the cell.
The present invention relates to a method of identifying specific linear and constrained discrete portions of pathogen antigens which either serve as epitopes for neutralizing antibodies or which are involved in pathogen binding to host cell receptors. By identifying discrete portions of pathogen antigens which are neutralizing epitopes, biologically active peptides can be constructed which are useful as components of vaccines against the pathogen. An effective neutralizing immune response will be elicited in a vaccinated individual. By identifying discrete portions of pathogen antigens which are involved in pathogen binding to host cell receptors, biologically active peptides can be constructed which are useful as agents which block pathogen attachment to cellular receptors. Additionally, by identifying discrete portions of pathogen antigens which are involved in pathogen binding to host cell receptors, biologically active peptides can be constructed which mimic pathogen antigens and act on mammalian cells by binding to the receptor sites of those cells to alter or affect their function or behavior, or which prevent or alter the effect which pathogen antigens would otherwise have upon those cells.
The present invention relates to the field of biologically active peptides which have some shared and/or similar amino acid sequences to the amino acid sequences of cellular receptor sites and thereby compete with such cellular receptors for binding to either biologically active proteins or pathogen antigens. In addition, the invention relates to the field of biologically active peptides which have some shared and/or similar amino acid sequences to the amino acid sequences of the ligand surface that attaches to a cellular receptor site. The ligand mimetic peptide can be used as a stimulant or inhibitor of that receptor. Where the biologically active peptide competes in pathogen/receptor binding, the biologically active peptides are useful to prevent pathogen attachment and thereby prevent infection. Where the biologically active peptide competes in biologically active protein/receptor binding, the biologically active peptides are useful to prevent ligand/receptor binding and thereby prevent the effect on cellular function or behavior normally associated with the biologically active protein/receptor binding.